Movement of the Escherichia Coli RNA Polymerase Clamp Modulates Intrinsic Transcription Termination PDF Download

Author: Michael John Allen Bellecourt
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Languages : en
Pages : 165

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Book Description
Cellular organisms use RNA polymerases (RNAPs) to express genes through the process of transcription. Accurate and timely RNA synthesis requires regulation of all steps of a complex transcription cycle, a regulatory framework involving the careful interplay of interactions between RNAP, the sequences and structure of template DNA and the nascent RNA, and myriad transcription factors that associate with both RNAP and the chromosome. At the end of a transcription unit, after kilobases of transcription, the elongation complex must efficiently release RNA and RNAP from DNA over a 2-3 nucleotide window. In bacteria, there are two programmed termination mechanisms. Intrinsic terminators utilize a GC-rich dyad immediately upstream to a U-rich tract that, together, promotes termination in the absence of additional transcription factors. Rho-dependent termination utilizes the RNA helicase Rho, which binds unstructured RNAs and utilizes its motor activity to extract the nascent RNA. Prior to my studies, the role of nucleic-acid sequence and structure in these mechanisms were well-characterized, but the role of protein conformation changes were largely unknown. To investigate how movements by the RNAP clamp affected intrinsic termination, I disentangled the steps of elongation, commitment, and dissociation, which are conflated in traditional termination measures. I found that clamp movements affected termination efficiency due to effects on elongation, not termination itself. Next, I found that clamp opening is necessary for release of RNAP from DNA, but not release of RNA. I also demonstrated that restricting movements of the trigger loop prior to commitment inhibits intrinsic termination, in agreement with a recently proposed multistate-multipath model of intrinsic termination. My studies also determined how the transcription factor NusG promotes Rho-dependent termination. After binding C-rich RNAs in its secondary site, the Rho ring makes small amino acid rearrangements in its C-terminal domain to induce ring closure. At subpar RNAs that Rho cannot effectively bind, NusG associates near this amino acid switch network, inducing the necessary rearrangements. My studies underscore the importance of protein movements during the termination process, from small-scale Brownian motion to large-scale movements of protein modules, and further demonstrate that RNAP might remain associated with DNA after termination.